Results and discussion

38 concentration of sulfate. The results for sulfate in plants before and after treatment were recorded.

2.3.7 Statistical data analysis

SPSS-Paired sample t-test is a data analysis method used to determine the statistical difference between two measurements or time points. It uses H₀ (null hypothesis) which states there is no difference if p< 0.05. Then H₀ is rejected. But if p>0.05, H₀ is accepted. The other hypothesis that is used by this test is the Hi (alternative hypothesis) which states that there is significant difference at a default significant level of 5% or 0.05. Paired sample t-test was used to analyze data in order to compare sulfate concentration within the plants harvested before and after exposure to sulfate contaminated water (treatment) and sulfate removal in the control and planted sections.

39 (Bidens pilosa L) were not negatively affected by toxic components of wastewater since they did not show any symptoms of growth inhibition.

2.4.2 Sulfate removal in the hydroponic system and the mechanisms of removal

Figure 8 shows sulfate removal efficiency over retention time (in hours) in the control and planted sections. Sulfate removal was increasing with the increasing hydraulic retention time both in the control and in the planted sections. There was a rapid increase of sulfate removal in the control section after 120 hours but it was increasing faster in the planted section compared to the control section. Sulfate removal in the control section was due to microbial degradation of sulfate by microorganisms that utilized sulfate. The rapid increase of sulfate removal in the planted section was after 96 hours. The plant uptake mechanism by Bidens pilosa L and microbial degradation of sulfate led to the rapid increase of sulfate removal in the planted section. According to Zhao et al. (2014) plant cells activity increased as the plants grew, leading to the increase in sulfate uptake by Bidens pilosa L. This is confirmed by Liu et al. (2017) who found that Bidens pilosa L had a potential to accumulate pollutants. The results of sulfate removal (Figure 8) proved that the aim of the study (which was to establish the effect of Bidens pilosa L in sulfate removal from industrial wastewater) was achieved. Bidens pilosa L positively influenced sulfate removal from wastewater. The highest removal efficiency was obtained after 288 hours in both systems (76% in the planted section) and (56%

in the control section).

40

T im e ( h )

Sulfate removal efficiency (%)

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2 2 1 6 2 4 0 2 6 4 2 8 8 0

1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0

P la n te d s e c tio n C o n tr o l s e c tio n

Figure 8: Sulfate removal efficiency (%) in the control and planted section over sampling periods.

After 288 hours, sulfate removal would be expected to stabilize due to the age of plants. It would also be expected that sulfate would be recycled back into water from plants and assimilated by plants again leading to the eventual wave of the sulfate removal. Similar findings were reported by (Scholz and Lee, 2005) who demonstrated that the results of sulfate removals were higher in planted section compared to the control section. The high removal percentages of sulfate in the planted section of the study that was conducted by (Scholz and Lee, 2005) was due to degradation of sulfate by microorganisms and macrophytes, which assisted in the reduction of sulfate from industrial wastewater through sulfate accumulation via the roots. Although other factors might have contributed in sulfate removal, Zhao et al.

(2014) has reported that vegetation played a major role in global recycling of persistent organic pollutants and their uptake from the environment into plant roots is a significant pathway. The results in Figure 8 support the findings by Zhao et al. (2014).

41 The results in Figure 8 support the fact that substrate (gravel and sand) and microorganisms all played a role in sulfate removal in both systems. The substrate served as a filter bed for sulfate salts within water since there was a removal of sulfate in the control section regardless of the absence of macrophytes but Bidens pilosa L also played a significant role in sulfate removal in the planted section.

In addition, sulfate removal in the hydroponic system after 240 hours seemed to reach a stationary phase (Figure 8). Aging of Bidens pilosa L macrophytes may have attributed to the static motion of sulfate assimilation. According to Kowalska, (2005), sulfate assimilation is dependent on the stage of plant growth. Young leaves have the most active sulfate assimilative reduction into organic compounds, the most intense sulfate assimilation is at the stage of maximal leaf growth. In the aging plants, organic sulfur decreases in leaves with the decrease in non-organic compounds which leads to the weakening of sulfate degradation processes and sulfate translocation to generative organs (Buchner et al., 2004). The competition between plants and microorganisms for sulfate might have led to the depletion of sulfate in water, which might also have been the reasonable explanation of the static mode of sulfate removal after 240 hours.

However, contradictory results were reported by Shamshad et al. (2016) where the steady state in sulfate removal was reached within 7 days due to acidic pH of wastewater resulting from high concentrations of sulfate. Acidic environment may have led to the inhibition of microalgae that was used in Shamshad’s et al. (2016) study since microalgae prefers environments with neutral or weakly alkaline pH. This proves that failure to choose macrophytes of choice that are able to withstand harsh or unfavourable conditions yields negative results. However, Bidens pilosa L used in this study was able to withstand harsh

42 condition. The macrophytes of choice removed sulfate without showing any symptoms of growth inhibition.

Table 1: Sulfate concentrations in wastewater over retention time (control section).

Time (h) pH Temperature (°C) Sulfate concentration

(mg/l)

0 5.03 22.0 705

24 5.35 21.7 684

48 6.15 23.6 659

72 6.17 24.1 620

96 6.22 26.2 590

120 6.34 25.1 577

144 6.1 23.4 515

168 6.6 22.9 460

192 6.9 22 409

216 7.4 23.1 370

240 6.5 24.6 330

264 6.4 21 316

288 5.8 20.8 309

The significance or the ability of macrophytes (Bidens pilosa L) in the wetland system to remove sulfate is supported by the sulfate concentrations shown in Tables 1 and 2. Even though microorganisms may have degraded sulfate in both systems, the presence of Bidens pilosa L was the cause of high sulfate removal in the planted section compared to the control section, since the final concentration in the planted section was 169 mg/l (<250 mg/l, the acceptable sulfate concentration in water) while in the control section it was 309 mg/l, which was way higher than 250 mg/l. This indicated that microorganisms played a role in sulfate degradation in the control section, but it was not as effective as in the planted section due to

43 the presence of Bidens pilosa L. Positive results in the study by Shamshad et al. (2016) were only achieved in the bioreactor with consortium of bacteria containing cyanobacteria and algae, until pH dropped to 4.4 and the steady stage was reached again. Shamshad et al. (2016) results proved that pH below 5 is unfavourable to the sulfate-degrading microorganisms.

Table 2: Sulfate concentrations in wastewater over retention time (planted section).

Time (h) pH Temperature (°C) Sulfate concentration

(mg/l)

0 5.03 22.0 705

24 6.61 26 670

48 6.3 24.2 630

72 6.5 25 585

96 6.0 21 526

120 7.2 25 460

144 7.0 23.8 390

168 6.8 22.4 316

192 6.5 21.6 256

216 6.8 23 201

240 6.0 20.9 190

264 5.42 24.7 184

288 5.85 21.6 169

The paired t-test was carried out in order to compare removal efficiency over hydraulic retention time between the control and planted sections. (P value=0.0001), p<0.05 and it was concluded that there was a significant difference between the removal of sulfate in the control and the planted section. Similar findings were reported by Kopriva et al. (2012).

44 2.4.2.1 Sulfate uptake by Bidens pilosa L

Figure 9 presents the results of sulfate concentration accumulated by Bidens pilosa L. Sulfate was present in all macrophytes but was in low concentrations in Bidens pilosa L harvested before treatment. Sulfate concentration in plants before treatment was 110 mg/l and this proved that sulfate was naturally present in the macrophytes in a form of sulfur (but not in excessive amounts) which was an essential element for plant growth. Sulfate concentrations increased in plants after treatment (exposure to sulfate contaminated water) and it was (353 mg/l). The increase in sulfate concentration in plants after treatment indicated that Bidens pilosa L had accumulated toxic sulfate, and assimilated this compound without any symptoms of stress. The high levels of sulfate present in Bidens pilosa L harvested after treatment was due to the assimilation of sulfate by the macrophytes via the roots. Kowalska (2005) stated that high content of sulfate in plants indicates intake of sulfate.

SO4 [mg/l]

Before treatm ent

After treatm ent 0

2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5 2 5 0 2 7 5 3 0 0 3 2 5 3 5 0 3 7 5 4 0 0

B e fo r e tr e a tm e n t A fte r tr e a tm e n t

Figure 9: Sulfate concentration in Bidens pilosa L harvested before and after treatment.

45 According to Sun (2009), excessive sulfate concentration in the root zone indicates excessive uptake by the plants roots which eventually leads to the increase in the synthesis of glutathione. The increase in glutathione synthesis tends to signal the decrease of sulfate intake. The paired t-test was also carried out in order to establish if there was a significant difference between the concentrations of sulfate in samples harvested before and after treatment. Since p<0.05 was less than the significant level, the null hypothesis was rejected, which means there was a significant difference between the concentrations of sulfate within plants that were harvested before and after treatment (Figure 9). Similar results were reported by Guittonny-Philipe et al. (2015). Bidens pilosa L and other macrophytes used in Guttonny’s study were tolerant to toxic sulfate and metals that they were exposed to. The increased sulfate concentration in macrophytes after treatment proved that the introduction of macrophytes in a wetland system had a positive effect on sulfate removal from mine water as documented by Zhao et al. (2014).